Waters Oligonucleotide Separation Technology ACQUITY UPLC C18 Column - Manual (Page 6)

6
[ method guidelines ]
1: Standard oligonucleotides: 10 – 30 mers
2: Short oligonucleotides: Less than 10 mer
3: Long oligonucleotides: 30 – 60 mers
4: Assuming use of a 2.1 x 50mm ACQUITY UPLC
® OST C
18
column
at a flow of 0.2 mL/min and a separation temperature of 60 ˚C.
The retention of dye labeled and dually-labeled oligonucleotides is
dictated by the nature of label. For example, the retention of 25 mer
oligonucleotide increases according to the type of label attached as
follows: no label<6FAM< <TAMRA<TET<HEX<Cy3.
VII. AnALYsIs oF ModIFIed oLIgonUCeUtIdes
ACQUITY UPLC ® OST C
18
columns are suitable for analysis of unmodi-
fied as well as modified detritylated oligodeoxyribonucleotides and
oligoribonucleotides. Phosphorothioate and/or 2’-O -alkyl modified
oligonucleotides can also be analyzed with IP-RP-HPLC method. The
modified oligonucleotides are usually more difficult to resolve from
N -1 etc. impurities. The recommended ion-pair system for phospho-
rothioate oligonucleotide analysis is TEA-HFIP (see Recommended
mobile phases).
Peptide nucleic acids (PNA) can also be analyzed using ACQUITY
UPLC
® OST C
18
columns. The ion-pairing system recommended for
analysis of PNA is similar to those used for peptide analysis (0.1%
Trifluoroacetic acid or formic acid).
Gradient 1
[standard oligonucleotides (1)]
Gradient 2
[High GC content or short oligonucleotides (2)]
Gradient 3
[High AT content or long oligonucleotides (3)]
Initial % ACN 7.00% 5.25% 7.50%
Final % ACN 10.75% 9.00% 12.50%
Gradient Length(4) 15 min 15 min 20 min
Table 2: Suggested Gradient Conditions for Non-standard Detritylated Synthetic Oligonucleotide Sequences
Waters Part Number Column Name Pore Size Particle Size Dimension
186003949 *ACQUITY UPLC ® OST C
18
135Å 1.7 micron 2.1 x 50 mm
186003950 *ACQUITY UPLC ® OST C
18
135Å 1.7 micron 2.1 x 100 mm
186003951 *Custom ACQUITY UPLC ® OST C
18
- - -
Table 3: Ordering Information
*Note: Optimum performance of ACQUITY UPLC ® OST C
18
columns is best assured using an appropriately configured Waters ACQUITY UPLC ® System.
Consequently, use of ACQUITY UPLC ® OST C
18
columns on conventional HPLC systems is not recommended.